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Article
SARS-CoV-2 Disrupts Splicing, Translation,
and Protein Trafficking to Suppress Host Defenses
Abhik K. Banerjee,1,2,8 Mario R. Blanco,1,8 Emily A. Bruce,3,8 Drew D. Honson,1,9 Linlin M. Chen,1,9 Amy Chow,1,9
Prashant Bhat,1,4 Noah Ollikainen,1 Sofia A. Quinodoz,1 Colin Loney,5 Jasmine Thai,1 Zachary D. Miller,6 Aaron E. Lin,7
Madaline M. Schmidt,3 Douglas G. Stewart,5 Daniel Goldfarb,5 Giuditta De Lorenzo,5 Suzannah J. Rihn,5
Rebecca M. Voorhees,1 Jason W. Botten,3 Devdoot Majumdar,6,* and Mitchell Guttman1,10,*
1Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA
2Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA
3Departments of Medicine, Division of Immunobiology and Microbiology, and Molecular Genetics, Larner College of Medicine, University of

Vermont, Burlington, VT 05405, USA

4David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA
5MRC-University of Glasgow Centre for Virus Research (CVR), Glasgow G61 1QH, UK
6Department of Surgery and University of Vermont Cancer Center, University of Vermont College of Medicine, 89 Beaumont Avenue,

Burlington, VT 05405, USA

7Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
8These authors contributed equally
9These authors contributed equally
10Lead Contact

*Correspondence: dev.Majumdar@med.uvm.edu (D.M.), mguttman@caltech.edu (M.G.)

https://doi.org/10.1016/j.cell.2020.10.004

SUMMARY

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently identified coronavirus that
causes the respiratory disease known as coronavirus disease 2019 (COVID-19). Despite the urgent need,
we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis. Here, we comprehensively
define the interactions between SARS-CoV-2 proteins and human RNAs. NSP16 binds to the mRNA recog-
nition domains of the U1 and U2 splicing RNAs and acts to suppress global mRNA splicing upon SARS-CoV-2
infection. NSP1 binds to 18S ribosomal RNA in the mRNA entry channel of the ribosome and leads to global
inhibition of mRNA translation upon infection. Finally, NSP8 and NSP9 bind to the 7SL RNA in the signal
recognition particle and interfere with protein trafficking to the cell membrane upon infection. Disruption of
each of these essential cellular functions acts to suppress the interferon response to viral infection. Our re-
sults uncover a multipronged strategy utilized by SARS-CoV-2 to antagonize essential cellular processes to
suppress host defenses.

INTRODUCTION 2020). These include 4 structural proteins: the nucleocapsid (N;

Coronaviruses are a family of viruses with notably large single-
stranded RNA genomes and broad species tropism among mam-
mals (Graham and Baric, 2010). Recently, a coronavirus, severe
acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was
discovered to cause the severe respiratory disease known as co-
ronavirus disease 2019 (COVID-19). It is highly transmissible in hu-
man populations, and its spread has resulted in a global pandemic
with more than a million deaths to date (Andersen et al., 2020; Zou
et al., 2020). We do not fully understand the molecular basis of
infection and pathogenesis of this virus in human cells. Accord-
ingly, there is an urgent need to understand these mechanisms
to guide the development of therapeutic agents.
SARS-CoV-2 encodes 27 proteins with diverse functional roles
in virus replication and packaging (Bar-On et al., 2020; Wang et al.,
which binds the viral RNA) and the envelope (E), membrane (M),
and spike (S) proteins, which are integral membrane proteins. In
addition, there are 16 non-structural proteins (NSP1–NSP16)
that encode the RNA-directed RNA polymerase, helicase, and
other components required for virus replication (da Silva et al.,
2020). Finally, there are 7 accessory proteins (ORF3a–ORF8)
whose function in virus replication or packaging remains largely
uncharacterized (Chen and Zhong, 2020; Finkel et al., 2020).
As obligate intracellular parasites, viruses require host cell
components to translate and transport their proteins and to
assemble and secrete viral particles (Maier et al., 2016). The
mammalian innate immune system acts to rapidly detect and
block viral infection at all stages of the virus life cycle (Chow
et al., 2018; Jensen and Thomsen, 2012; Wilkins and Gale,
2010). The primary form of intracellular virus surveillance

Cell 183, 1325–1339, November 25, 2020 ª 2020 The Authors. Published by Elsevier Inc. 1325
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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engages the interferon pathway, which amplifies signals result-
ing from detection of intracellular viral components to induce a
systemic type I interferon response upon infection (Stetson
and Medzhitov, 2006). Specifically, cells contain various RNA
sensors (such as RIG-I and MDA5) that detect the presence of
viral RNAs and promote nuclear translocation of the transcription
factor IRF3, leading to transcription, translation, and secretion of
interferon (e.g., interferon [IFN]-a and IFN-b). Binding of IFN to
cognate cell-surface receptors leads to transcription and trans-
lation of hundreds of antiviral genes.
In order to successfully replicate, viruses employ a range of
strategies to counter host antiviral responses (Beachboard and
Horner, 2016). In addition to their essential roles in the viral life
cycle, many viral proteins also antagonize core cellular functions
in human cells to evade host immune responses. For example,
human cytomegalovirus (HCMV) encodes proteins that inhibit
major histocompatibility complex (MHC) class 1 display on the
cell surface by retaining MHC proteins in the endoplasmic retic-
ulum (Miller et al., 1998), polioviruses encode proteins that
degrade translation initiation factors (eIF4G) to prevent transla-
tion of 50 -capped host mRNAs (Kempf and Barton, 2008; Lloyd,
2006), and influenza A encodes a protein that modulates mRNA
splicing to degrade the mRNA that encodes RIG-I (Kochs et al.,
2007; Zhang et al., 2018).
Suppression of the IFN response has recently emerged as a
major clinical determinant of COVID-19 severity (Zhang et al.,
2020), with almost complete loss of secreted IFN characterizing
the most severe cases (Hadjadj et al., 2020). The extent to which
SARS-CoV-2 suppresses the IFN response is a key character-
istic that distinguishes COVID-19 from SARS and Middle East
respiratory syndrome (MERS) (Lokugamage et al., 2020). Several
strategies have been proposed for how the related SARS- and
MERS-causing viruses may hijack host cell machinery and
evade immune detection, including repression of host mRNA
transcription in the nucleus (Canton et al., 2018), degradation
of host mRNA in the nucleus and cytoplasm (Kamitani et al.,
2009; Lokugamage et al., 2015), and inhibition of host translation
(Nakagawa et al., 2018). Nonetheless, the extent to which SARS-
CoV-2 uses these or other strategies and how they may be
executed at a molecular level remains unclear.
Understanding the interactions between viral proteins and
components of human cells is essential for elucidating their path-
ogenic mechanisms and for development of effective therapeu-
tic agents. Because SARS-CoV-2 is an RNA virus, and many of
its encoded proteins are known to bind RNA (Sola et al., 2011),
we reasoned that these viral proteins may interact with specific
human mRNAs (critical intermediates in protein production) or
non-coding RNAs (critical structural components of diverse
cellular machines) to promote virus propagation.
Here we comprehensively define the interactions between
each SARS-CoV-2 protein and human RNA. We show that 10
viral proteins form highly specific interactions with mRNAs or
noncoding RNAs (ncRNAs), including those involved in progres-
sive steps of host cell protein production. We show that NSP16
binds to the mRNA recognition domains of the U1 and U2 RNA
components of the spliceosome and acts to suppress global
mRNA splicing in SARS-CoV-2-infected human cells. We find
that NSP1 binds to a precise region on the 18S ribosomal RNA
1326 Cell 183, 1325–1339, November 25, 2020
Article
that resides in the mRNA entry channel of the initiating 40S ribo-
some. This interaction leads to global inhibition of mRNA trans-
lation upon SARS-CoV-2 infection of human cells. Finally, we
find that NSP8 and NSP9 bind to discrete regions on the 7SL
RNA component of the signal recognition particle (SRP) and
interfere with protein trafficking to the cell membrane upon infec-
tion. We show that disruption of each of these essential cellular
functions acts to suppress the type I IFN response to viral infec-
tion. Our results uncover a multipronged strategy utilized by
SARS-CoV-2 to antagonize essential cellular processes and
robustly suppress host immune defenses.
RESULTS
Comprehensive Mapping of SARS-CoV-2 Protein
Binding to Human RNAs
We cloned all 27 of the known SARS-CoV-2 viral proteins into
mammalian expression vectors containing an N-terminal Halo-
Tag (Los et al., 2008; Figure S1A; STAR Methods), expressed
each in HEK293T cells, and exposed them to UV light to cova-
lently crosslink proteins to their bound RNAs. We then lysed
the cells and purified each viral protein using stringent dena-
turing conditions to disrupt any non-covalent associations and
capture those with a UV-mediated interaction (Figure 1A; STAR
Methods). As positive and negative controls, we purified a known
human RNA binding protein (PTBP1) and a metabolic protein
(GAPDH) (Figures S1A–S1E).
We successfully purified 26 of the 27 viral proteins (Figure S1A;
full-length S was not soluble when expressed). We found that 10
viral proteins (NSP1, NSP4, NSP8, NSP9, NSP12, NSP15,
NSP16, ORF3b, N, and E) bind to specific host RNAs (p <
0.001; Figure 1B; Table S1), including 6 structural ncRNAs and
142 mRNAs (Table S1). These include mRNAs involved in protein
translation (e.g., COPS5, EIF1, and RPS12,), protein transport
(ATP6V1G1, SLC25A6, and TOMM20), protein folding (HSPA5,
HSPA6, and HSPA1B), transcriptional regulation (YY1, ID4, and
IER5), and immune response (JUN, AEN, and RACK1) (false dis-
covery rate [FDR] < 0.05; Figures 1B and S1F). Importantly, the
observed interactions are highly specific for each viral protein,
and each protein binds to a precise region within each RNA (Fig-
ures 1C and S1F).
Using these data, we identified several viral proteins that
interact with structural ncRNA components of the spliceosome
(U1 and U2 small nuclear RNA [snRNA]), the ribosome (18S
and 28S rRNA), and the SRP (7SL) (Figure 1B). Because these
molecular machines are essential for three essential steps of
protein production—mRNA splicing, translation, and protein
trafficking—we focused on their interactions with viral proteins
to understand their functions and mechanisms in SARS-CoV-2
pathogenesis.
NSP16 Binds to the Pre-mRNA Recognition Domains of
the U1 and U2 snRNAs
After transcription in the nucleus, nascent pre-mRNAs are spliced
to generate mature mRNAs that are translated into protein.
Splicing is mediated by a complex of ncRNAs and proteins known
as the spliceosome. Specifically, the U1 snRNA hybridizes to the 50
splice site at the exon-intron junction, and the U2 snRNA

Article
A B
Figure 1. Global RNA Binding Maps of SARS-CoV-2 Proteins
(A) Schematic of our approach.
(B) Enrichment heatmap of each SARS-CoV-2 protein (rows) by significantly enriched 100-nt RNA bins (columns; p < 0.001 and enrichment > 3-fold; STAR
Methods). Shared colored bars indicate multiple bins within the same mRNA. For spacing reasons, the 82 mRNAs bound by N protein are displayed separately.
(C) Examples of sequencing reads over specific mRNAs for viral proteins (red) relative to input RNA coverage (gray) are shown. Coding regions (thick lines) and
untranslated regions (thin lines) are shown for each mRNA.
See also Table S1.
hybridizes to the branchpoint site in the intron to initiate splicing of
virtually all human mRNAs (Séraphin et al., 1988).
We identified a highly specific interaction between the NSP16
viral protein and the U1 and U2 snRNAs (Figure 1B). Because U1
and U2 are small RNAs (164 and 188 nt, respectively), we noticed
strong enrichment of NSP16-associated reads across the entire
length of each. To more precisely define the binding sites, we ex-
ploited the well-described tendency of reverse transcriptase to
preferentially terminate when it encounters a UV-crosslinked pro-
tein on RNA (König et al., 2010; Figures 1A and S1D). We deter-
mined that NSP16 binds to the 50 splice site recognition sequence
of U1 (Figures 2A, 2B, S2A, and S2B) and the branchpoint recog-
nition site of U2 (Figures 2C, 2D, S2C, and S2D). These binding
sites are highly specific to NSP16 relative to all of the other viral
and human proteins (Figures 1B, S2A, and 2C). Consistent with
its interaction with U1/U2, we observed that NSP16 localizes in
the nucleus upon SARS-CoV-2 infection (Figures 2E, S2E, and
S2F) and when expressed in human cells (Figure S2G).
NSP16 Disrupts Global mRNA Splicing upon SARS-CoV-
2 Infection
Based on the locations of the NSP16 binding sites relative to the
mRNA recognition domains of the U1/U2 spliceosomal compo-
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nents, we hypothesized that NSP16 might disrupt splicing of
newly transcribed genes (Figure 2F). To test this, we co-ex-
pressed NSP16 in human cells along with a splicing reporter
derived from IRF7 (an exon-intron-exon minigene) fused to
GFP (Majumdar et al., 2018). In this system, if the reporter is
spliced, then GFP is made; if not, then translation is terminated
(via a stop codon present in the first intron), and GFP is not pro-
duced (Figure 3A). We observed a more than 3-fold reduction in
GFP levels in the presence of NSP16 compared with a control
human protein (Figures 3B and S3A).
To explore whether NSP16 has a global effect on splicing of
endogenous mRNAs, we measured the splicing ratio of each
gene using nascent RNA sequencing. Specifically, we metaboli-
cally labeled nascent RNA by feeding cells for 20 min with 5-ethy-
nyl uridine (5EU), purified and sequenced 5EU-labeled RNA, and
quantified the proportion of unspliced fragments spanning the 30
splice site of each gene (Figures 3C and S3B). We observed a
global increase in the fraction of unspliced genes in the presence
of NSP16 compared with controls (Figures 3D, S3C, and S3D).
Given that NSP16 is sufficient to suppress global mRNA
splicing, we expect that its expression in SARS-CoV-2-infected
cells would result in a global mRNA splicing deficit. To test this,
we infected human lung epithelial cells (Calu3) with SARS-CoV-2
Cell 183, 1325–1339, November 25, 2020 1327
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A B

C
D

E F

Figure 2. NSP16 Binds to U1 and U2 at Their mRNA Recognition Sites

(A) NSP16 enrichment of reverse transcription stop positions across each nucleotide of U1 (red) compared with a control protein (GAPDH, black). The red box
(below the x axis) represents most enriched nucleotide positions (U1, 9–13 nt). The gray-shaded box (overlay) outlines the position of the splice site recognition
sequence.
(B) Left: structure of the pre-catalytic human spliceosome (PDB: 6QX9; Charenton et al., 2019), highlighting the location of NSP16 binding site (red spheres)
relative to U1 (yellow ribbon) and mRNA (purple ribbon). Right: schematic of the structure.
(C) Enrichment across each nucleotide of U2 for NSP16 (red) and GAPDH (black). The red box demarcates most enriched nucleotide positions (U2, 27–34 nt). The
gray-shaded box outlines the location of the branchpoint recognition sequence.
(D) Structure of the pre-catalytic human spliceosome (PDB: 6QX9; Charenton et al., 2019) displaying the NSP16 binding site (red spheres), U2 (orange), and
mRNA (purple).
(E) Mock-infected (top) or SARS-CoV-2 infected (bottom) Vero E6 cells immunostained with a polyclonal antibody to NSP16 (left) or NSP1 (right). Imaris 3D
reconstruction of the DAPI (nucleus) and NSP16 or NSP1 signal are shown for each protein. The signal contained within the 3D nuclear volume (blue) is shown in
yellow and the cytoplasmic signal in purple. Scale bars, 3 mm.
(F) Model: NSP16 binding to U1/U2 can affect mRNA recognition during splicing.

and measured the splicing levels of newly transcribed mRNAs
compared with a mock-infected control. As expected, we
observed a global increase in the fraction of unspliced tran-
scripts upon SARS-CoV-2 infection, with 90% of measured
genes showing increased intron retention (Figures 3E and S3E).
These results indicate that NSP16 binds to the splice site and
branchpoint sites of U1/U2 to suppress global mRNA splicing in
SARS-CoV-2-infected cells (Figure 3F). Although NSP16 is known
to act as an enzyme that deposits 20 -O-methyl modifications on
viral RNAs (Decroly et al., 2011), our results demonstrate that it
also acts as a host virulence factor. Global disruption of mRNA
splicing may act to decrease host protein and mRNA levels by trig-
gering nonsense-mediated decay of improperly spliced mRNAs
(Kurosaki et al., 2019). Consistent with this, we observed a strong
global decrease in steady-state mRNA levels (relative to ncRNA
levels) upon SARS-CoV-2 infection (Figure S3F).

1328 Cell 183, 1325–1339, November 25, 2020

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A B

D E

F G H

Figure 3. NSP16 Suppresses Host mRNA Splicing

(A) Schematic of fluorescence reporter used to assay mRNA splicing.
(B) GFP density plot of HEK293T cells expressing the GFP splicing reporter and either GAPDH (gray) or NSP16 (red).
(C) Schematic of the nascent RNA purification method.
(D) The percentage of unspliced difference for each gene between HEK293T cells transfected with GAPDH (gray) or NSP16 (red). The plot represents the merge of
four independent biological replicates; replicates are plotted in Figure S4C.
(E) Violin plot for SARS-CoV-2 infected human lung epithelial cells (MOI = 0.01, 48 h) compared with mock infection. Plots are merges of two biological replicates;
replicates are plotted in Figure S4E.
(F) Model. NSP16 binding to U1 and U2 can reduce overall mRNA and protein levels.
(G) Expression of an IFN-stimulated gene (ISG) reporter upon transfection with GAPDH (gray) or NSP16 (red) after stimulation with IFN-b. Three independent
biological replicates; **p < 0.01.
(H) Example of nascent RNA sequencing at the intron of ISG15 (intron, line; exon, box) upon SARS-CoV-2 (red) or mock (gray) infection.

Inhibition of mRNA Splicing Suppresses the Host IFN
Response to Viral Infection
Because many of the key genes stimulated by IFN are spliced,
we reasoned that mRNA splicing would be critical for a robust
IFN response. To test this, we utilized a reporter line engineered
to express alkaline phosphatase upon IFN signaling (mimicking
an antiviral response gene). This IFN-stimulated gene (ISG) re-
porter line can be stimulated using IFN-b and assayed for re-
porter induction. We observed strong repression of this IFN-
responsive gene upon expression of NSP16 (Figure 3G) and
upon addition of a small molecule that interferes with spliceoso-
mal assembly (Figure S3G). These results demonstrate that one

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A B

C D

E F G

H I J

Figure 4. NSP1 Binds to 18S Near the mRNA Entry Channel to Suppress Translation
(A) NSP1 enrichment across each nucleotide of 18S. The cyan box indicates the most enriched nucleotides of NSP1 binding (18S, 607–644 nt).
(B) The location of NSP1 binding (cyan spheres) relative to the known structure of 40S (gray) and mRNA (purple ribbon). Right: schematic illustrating structure
(Ameismeier et al., 2018) and how NSP1 binding would block mRNA entry.
(C) Images of HEK293T cells co-expressing the GFP reporter and GAPDH (top) or NSP1 (bottom).
(D) Flow cytometry quantification (mean intensity) of GFP in the presence of GAPDH, NSP8/9, M, or NSP1 proteins. Three independent biological replicates per
condition.
(E) Puromycin incorporation (top) or total actin levels (bottom) measured in Calu3 cells infected with SARS-CoV-2 (MOI = 0.01, 48 h) or a mock-infected control
(left 2 lanes).
(F) The ratio of puromycin signal over total actin signal is plotted for each individual replicate.
(G) Read enrichment on 18S for an independent replicate of NSP1 wild type, NSP1 R124A/K125A mutant, and NSP1 K164A/H165A (DRC) mutant.
(H) Flow cytometry analysis of HEK293T cells transfected with GFP and NSP1DRC mutant (gray), wild-type NSP1, or NSP1 R124A/K125A (cyan).

(legend continued on next page)

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Article
outcome of NSP16-mediated inhibition of mRNA splicing is to
reduce the host cells’ innate immune response to virus recogni-
tion. Consistent with such a role, we observed an increase in
intron retention in multiple IFN-responsive genes (such as
ISG15 and RIG-I) upon SARS-CoV-2 infection (Figures 3H,
S3H, and S3I).
NSP1 Binds to 18S Ribosomal RNA in the mRNA Entry
Channel of the 40S Subunit
When exported to the cytoplasm, spliced mRNA is translated
into protein on the ribosome. Initiation of translation begins
with recognition of the 50 cap by the small 40S subunit (which
scans the mRNA to find the first start codon). We observed
that NSP1 binds exclusively to the 18S ribosomal RNA (Figures
1B and S4A)—the structural RNA component of the 40S ribo-
somal subunit.
Several roles of NSP1 have been reported in SARS-CoV and
MERS-CoV, including roles in viral replication, translational inhi-
bition, transcriptional inhibition, mRNA degradation, and cell cy-
cle arrest (Brockway and Denison, 2005; Kamitani et al., 2009;
Lokugamage et al., 2015; Narayanan et al., 2015). One of the re-
ported roles of NSP1 in SARS-CoV is that it can associate with
the 40S ribosome to inhibit host mRNA translation (Kamitani
et al., 2009; Tanaka et al., 2012), but it remains unknown whether
this association is due to interaction with the ribosomal RNA,
protein components of the ribosome, or other auxiliary ribosomal
factors. Accordingly, the mechanisms by which NSP1 acts to
suppress protein production remain elusive.
We mapped the location of NSP1 binding to a 37-nt region
corresponding to helix 18 (Figure 4A), adjacent to the mRNA en-
try channel (Simonetti et al., 2020; Figure 4B). The interaction
would position NSP1 to disrupt 40S mRNA scanning and prevent
translation initiation (Figure 4B) and disrupt tRNA recruitment to
the 80S ribosome and block protein production (Figure S4B).
Interestingly, the NSP1 binding site includes the highly
conserved G626 nucleotide, which monitors the minor groove
of the codon-anticodon helix for tRNA binding fidelity (Ogle
et al., 2001). We noticed that the C-terminal region of NSP1
has structural regions similar to SERBP1 (Brown et al., 2018)
and Stm1 (Ben-Shem et al., 2011), two known ribosome inhibi-
tors that bind in the mRNA entry channel to preclude mRNA ac-
cess (Figure S4C). Consistent with this, a recent cryo-EM struc-
ture confirms that NSP1 binds to these same nucleotides of 18S
within the mRNA entry channel (Thoms et al., 2020).
NSP1 Suppresses Global Translation of Host mRNAs
upon SARS-CoV-2 Infection
Given the location of NSP1 binding on the 40S ribosome, we hy-
pothesized that it could suppress global initiation of mRNA trans-
lation. To test this, we performed in vitro translation assays of a
GFP reporter in HeLa cell lysates and found that addition of
NSP1 led to potent inhibition of translation (Figure S4D). We
observed a similar NSP1-mediated translational repression when
we co-expressed NSP1 and a GFP reporter gene in HEK293T cells
(I) Quantification of the IFN-b response in the presence of GAPDH (gray) or NSP1 (cyan).
(J) Schematic of how NSP1 acts to suppress mRNA translation.
Error bars represent standard deviation across biological replicates, and dots represent individual values for each replicate; *p < 0.05 and **p < 0.01.
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(Figures 4C and 4D). In contrast, we did not observe this inhibition
when we expressed other SARS-CoV-2 proteins (NSP8, NSP9, or
M) or human proteins (GAPDH) (Figure 4D).
To determine whether NSP1 leads to translational inhibition of
endogenous proteins in human cells, we used a technique called
surface sensing of translation (SUnSET) to measure global pro-
tein production levels (Schmidt et al., 2009). In this assay, trans-
lational activity is measured by the level of puromycin incorpora-
tion into elongating polypeptides (Figure S4E). We observed a
strong reduction in the level of global puromycin integration in
cells expressing NSP1 compared with cells expressing GFP (Fig-
ures S4F and S4G).
Because NSP1 expression is sufficient to suppress global
mRNA translation in human cells, we hypothesized that SARS-
CoV-2 infection would also suppress global translation. To test
this, we infected a human lung epithelial (Calu3) or monkey kid-
ney (Vero) cell line with SARS-CoV-2 and measured nascent pro-
tein synthesis levels using SUnSET. We observed a strong
reduction of global puromycin integration upon SARS-CoV-2
infection in both cell types (Figures 4E, 4F, S4H, and S4I).
To explore whether NSP1 binding to 18S rRNA is critical for
translational repression, we generated a mutant NSP1 in which
two positively charged amino acids (K164 and H165) in the C-ter-
minal domain were replaced with alanine residues (Figure S4C;
Narayanan et al., 2008). We observed complete loss of in vivo con-
tacts with 18S (Figure 4G); because this mutant disrupts ribosome
contact, we refer to it as NSP1DRC. We co-expressed GFP and
NSP1DRC in HEK293T cells and found that the mutant fails to
inhibit translation (Figures 4H and S4J). In contrast, mutations to
the positively charged amino acids at positions 124/125 do not
affect 18S binding (Figure 4G) or the ability to inhibit translation
(Figure 4H).
These results demonstrate that NSP1 binds in the mRNA entry
channel of the ribosome and that this interaction is required for
translational inhibition of host mRNAs upon SARS-CoV-2
infection.
NSP1-Mediated Translational Inhibition Suppresses the
Host IFN Response
We explored whether NSP1 binding to 18S rRNA suppresses the
ability of cells to respond to IFN-b stimulation upon viral infec-
tion. We transfected ISG reporter cells with NSP1, stimulated
with IFN-b, and observed robust repression of the IFN-respon-
sive gene (>6-fold; Figure 4I). To confirm that this NSP1-medi-
ated repression occurs in human cells upon activation of dou-
ble-stranded RNA (dsRNA)-sensing pathways typically
triggered by viral infection, we treated a human lung epithelial
cell line (A549) with poly(I:C), a molecule that is structurally
similar to dsRNA and known to induce an antiviral innate immune
response (Alexopoulou et al., 2001; Kato et al., 2006) (Fig-
ure S4K). We observed marked downregulation of IFN-b protein
and endogenous IFN-b-responsive mRNAs in the presence of
NSP1 but not in the presence of NSP1DRC (Figures S4L and
S4M). These results demonstrate that NSP1, through its
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A B C

D E F

Figure 5. The 50 Viral Leader Protects mRNA from NSP1-Mediated Translational Inhibition
(A) Images of cells co-transfected with NSP1 and mCherry alone ( leader, top) or mCherry fused to the SARS-CoV-2 leader (+ leader, bottom).
(B) GFP (green) or mCherry (red) levels when fused to the viral leader (+ leader, right) or lacking the viral leader ( leader, left).
(C) GFP reporter with no leader (left), full leader (center), or stem loop 1 (SL1) upon NSP1 expression.
(D) Calu3 cells expressing SL1 fused to GFP. Cells were mock or SARS-CoV-2 infected (MOI = 0.1), and GFP expression was measured 24 h after infection by flow
cytometry.
(E) GFP reporter containing SL1 (left), a swap of SL2 and SL1 (SL2-SL1), insertion of 5 nt between the 50 end and SL1 (+5 nt-SL1), or no leader. GFP protein level
was measured for each condition upon expression of NSP1.
(F) Proposed model of how NSP1 binding to the viral leader can allosterically modulate NSP1 structure to protect mRNAs in cis.
Error bars represent standard deviation across biological replicates, and dots represent individual replicate values; *p < 0.05 and **p < 0.01.

interaction with 18S rRNA, suppresses the innate immune
response to virus recognition (Figure 4J).
The Viral 50 Leader Protects mRNA from NSP1-Mediated
Translational Inhibition
Because NSP1 blocking the mRNA entry channel would affect
host and viral mRNA translation, we explored how translation
of viral mRNAs is protected from NSP1-mediated translational
inhibition. Many viruses contain 50 untranslated regions that
regulate viral gene expression and translation (Gaglia et al.,
2012); all SARS-CoV-2-encoded subgenomic RNAs contain a
common 50 leader sequence that is added during negative-
strand synthesis (Kim et al., 2020b). We explored whether the
leader sequence protects viral mRNAs from translational inhibi-
tion by fusing the viral leader sequence to the 50 end of GFP or
mCherry reporter genes (Figure S5A). We found that NSP1 fails
to suppress translation of these leader-containing mRNAs (Fig-
ures 5A, 5B, and S5B). We dissected the leader sequence and
found that the first stem loop (SL1) is sufficient to prevent trans-
lational suppression upon NSP1 expression (Figure 5C) or
SARS-CoV-2 infection (Figure 5D).
We considered three models for how the leader could protect
viral mRNAs: (1) it could compete with the ribosome for NSP1 bind-
ing, (2) it could directly recruit free ribosomes, or (3) NSP1 could
bind to the leader independent of its ribosome interaction to alloste-
rically modulate the NSP1-ribosome interaction. We reasoned that
if the leader competes for NSP1 binding or directly recruits free ri-
bosomes, then the presence of SL1 should be sufficient for protec-
tion, regardless of its precise position in the 50 UTR. In contrast, if
the leader allosterically modulates ribosome binding, then the
spacing between the 50 cap (which is bound to NSP1-40S) and
SL1 would be critical for protection. To distinguish between these
models, we swapped the location of SL1 and SL2 in the 50 leader
or inserted 5 nt between the 50 cap and SL1 (Figure S5C) and found
that both mutants ablate protection (Figures 5E and S5D).
These results indicate that an mRNA requires the 50 leader to
be precisely positioned relative to the NSP1-bound 40S ribo-
some to enable translational initiation (Figure 5F). Although
many aspects of this allosteric model remain to be explored, it
would explain how leader-mediated protection can occur on
an mRNA only when present in cis. Moreover, this model sug-
gests that NSP1 might also act to further increase viral mRNA
translation by actively recruiting the ribosome to its own mRNAs.
Consistent with this, we observed a consistent, 20% increase
in translation of leader-containing reporter levels upon viral infec-
tion (Figure 5D) or expression of NSP1 (Figure S5E).

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A B

Figure 6. NSP8 and NSP9 Bind to 7SL RNA of the SRP

(A) Enrichment of reverse transcription stop positions across each nucleotide of 7SL is shown for NSP8 (blue) and NSP9 (red). Red (7SL, 142–143 nt; 7SL, 149–
151 nt) and blue (7SL, 193–194 nt) boxes demarcate the most enriched nucleotide positions.
(B) The locations of the NSP8 (blue spheres) and NSP9 (red spheres) binding sites on the S domain of 7SL (yellow ribbon) structure relative to SRP54 and SRP19
(gray) (PDB: 1MFQ; Kuglstatter et al., 2002). Right: schematic of the structure and model of how NSP8/9 binding to 7SL could affect SRP protein binding.
(C) Read enrichment across each nucleotide of 28S for NSP8 (blue). The black box indicates the location of the ES27 expansion sequence (28S, 2,889–3,551 nt).
The blue box indicates the most enriched nucleotide position on 28S rRNA (28S, 3,017–3,529 nt).
(D) The locations of the NSP8 (blue) and NSP9 (red) binding sites relative to the structure of the SRP-ribosome complex (PDB: 3JAJ; Voorhees and Hegde, 2015)
superimposed on the structure of the ES27 region of 28S (Ebp1-ribosome complex; PDB: 6SXO; Wild et al., 2020). The observed NSP8 binding site in the ES27
region of 28S (gray) is demarcated in blue, and the NSP8 (blue) and NSP9 (red) binding sites on 7SL (yellow) are highlighted. Right: schematic illustrating the
interaction between the ribosome and SRP.

NSP8 and NSP9 Bind to the 7SL RNA Component of SRP
Upon engaging the start codon in an mRNA, the 60S subunit of
the ribosome is recruited to form the 80S ribosome, which trans-
lates mRNA. SRP is a universally conserved complex that binds
to the 80S ribosome and acts to co-translationally scan the
nascent peptide to identify hydrophobic signal peptides present
in integral membrane proteins and proteins secreted from the
plasma membrane (Akopian et al., 2013). When these are identi-
fied, SRP triggers ribosome translocation to the endoplasmic re-
ticulum (ER) to ensure proper folding and trafficking of these pro-
teins to the cell membrane (Akopian et al., 2013).
We identified two viral proteins, NSP8 and NSP9, that bind at
distinct and highly specific regions in the S domain of the 7SL
RNA scaffold of SRP (Figures 6A and S6A). NSP8 interacts
with 7SL in the region bound by SRP54 (the protein responsible
for signal peptide recognition, SRP-receptor binding, and ribo-
some translocation; Akopian et al., 2013; Holtkamp et al.,
2012; Figure 6B). NSP9 binds to 7SL in the region that is bound
by the SRP19 protein (Figure 6B), which is required for proper
folding and assembly of the SRP (including proper loading of
SRP54; Akopian et al., 2013).
Because SRP scans nascent peptides co-translationally, we
were intrigued to find that NSP8 also forms a highly specific
interaction with 28S rRNA (the structural component of the 60S
subunit) (Figures 6C and S6B). The binding site on 28S rRNA cor-
responds to the largest human-specific expansion segment in
the ribosome, referred to as ES27 (Parker et al., 2018). ES27 is
highly dynamic and, thus, has not been resolved in most ribo-
some structures (Zhang et al., 2014). However, when engaged
by specific factors, ES27 can become ordered and has been

Cell 183, 1325–1339, November 25, 2020 1333

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shown recently to be capable of interacting with the ribosome
exit tunnel adjacent to the 60S binding site of SRP (Figures 6D
and S6C; Wild et al., 2020).
These observations suggest that NSP8 and NSP9 bind to the
co-translational SRP complex. Consistent with this, we find that
NSP8 and NSP9 localize broadly throughout the cytoplasm
when expressed in human cells (Figure S6D) or upon SARS-
CoV-2 infection (Figures S6E and S6F).
NSP8 and NSP9 Suppress Protein Integration into the
Cell Membrane
Because NSP8 and NSP9 binding on 7SL are positioned to
disrupt SRP function, we hypothesized that they may alter trans-
location of secreted and integral membrane proteins
(Figure S7A).
To test this, we expressed an SRP-dependent membrane pro-
tein (nerve growth factor receptor [NGFR]; Izon et al., 2001) fused
via an internal ribosome entry site (IRES) to a non-membrane
GFP (Figure S7F). In this system, when a perturbation specifically
affects membrane protein levels, we expect to see a decrease in
the ratio of membrane to non-membrane protein levels. To
ensure that the NGFR reporter accurately reports SRP function,
we treated HEK293T cells with small interfering RNAs (siRNAs)
against SRP54 or SRP19 and found that both lead to a dramatic
reduction of the NGFR membrane protein relative to the non-
membrane GFP protein (Figure S7B). Similarly, we found that
expression of NSP8 and NSP9 (alone or together) led to a striking
reduction in expression of NGFR relative to GFP (Figure 7A).
Expression of control proteins did not specifically affect NGFR
levels (Figures 7A and S7B).
To determine whether there is a global effect on membrane
protein levels, we utilized the SUnSET method to measure puro-
mycin levels in membrane proteins using flow cytometry (STAR
Methods). We confirmed that disruption of SRP leads to a global
reduction in puromycin levels in the cell membrane (Figure S7C).
We observed a comparable global reduction of puromycin-
labeled membrane proteins upon expression of NSP8 or NSP9
individually or together but not with control proteins (Figures
7B and S7C).
SARS-CoV-2 Infection Suppresses Protein Integration
into the Cell Membrane
Because NSP8 and NSP9 are each sufficient to suppress protein
integration into the cell membrane, we anticipated that SARS-
CoV-2 infection would lead to similar suppression. However,
determining whether SARS-CoV-2 infection specifically affects
membrane protein expression is confounded by the fact that
NSP1 inhibits translation of membrane and non-membrane pro-
teins upon infection.
To address this, we co-expressed a membrane protein re-
porter (NGFR) containing the 50 viral leader along with a non-
membrane GFP reporter containing the viral leader. Upon viral
infection, we observed a strong reduction in membrane protein
levels (Figure 7C) but no reduction in non-membrane GFP levels
(Figure 5D). To ensure that these effects are specific to SARS-
CoV-2-infected cells, we separated individual cells in the in-
fected population into those expressing the viral S protein (S+)
and those not expressing the protein (S). We found that the
1334 Cell 183, 1325–1339, November 25, 2020
Article
shift in membrane protein levels only occurred in S+ cells (Fig-
ure 7D), whereas the S- population resembled the mock-infected
samples (Figure 7C). We observed a strong relationship between
the level of S protein, likely reflecting the amount of viral replica-
tion in each cell, and the level of membrane protein suppression
(Figure 7C). We observed this membrane protein-specific
decrease upon infection of human lung epithelial cells (Calu3;
Figure S7D) and monkey kidney cells (Vero; Figures 7C and 7D).
These results demonstrate that NSP8 and NSP9 bind to 7SL to
disrupt SRP function and suppress membrane protein trafficking
in SARS-CoV-2-infected cells. Although NSP8 and NSP9 are
thought to be components of the virus replication machinery
(Sutton et al., 2004), our results indicate that they play an addi-
tional role as host virulence factors. Because viral membrane
proteins also require trafficking to the ER, viral disruption of
SRP might also negatively impact virus propagation, unless viral
proteins are trafficked in an SRP-independent manner (Fig-
ure S7E) or if NSP8/9 selectively affects host (but not viral) pro-
tein trafficking.
Viral Disruption of Protein Trafficking Suppresses the
IFN Response
Next we explored how disruption of SRP might be advantageous
for virus propagation. Because secretion of IFN and other cyto-
kines is dependent on the SRP complex for secretion (Fig-
ure S7F), a central component of the IFN response is dependent
on SRP. Accordingly, we hypothesized that NSP8/9-mediated
viral suppression of SRP would act to suppress the IFN response
upon infection. To test this, we co-expressed NSP8 and NSP9
and observed a significant reduction in the IFN response relative
to a control protein (Figure S7G).
These results suggest that SARS-CoV-2-mediated suppres-
sion of SRP-dependent protein secretion enables suppression
of host immune defenses (Figure 7E). Interestingly, many pro-
teins involved in anti-viral immunity, including most cytokines
and MHC class I, are membrane anchored or secreted and are
known to use the SRP pathway for transport (Vermeire et al.,
2014; Figure S7F), suggesting that there may be other effects
of SRP pathway inhibition on SARS-CoV-2 pathogenesis.
DISCUSSION
We identified several pathogenic functions of SARS-CoV-2 in hu-
man cells, including global inhibition of host mRNA splicing, pro-
tein translation, and membrane protein trafficking, and described
the molecular mechanisms by which the virus acts to disrupt these
essential cell processes. Interestingly, all of the viral proteins
involved (NSP1, NSP8, NSP9, and NSP16) are produced in the
first stage of the virus life cycle prior to generation of dsRNA prod-
ucts during virus genome replication. Because dsRNA is detected
by host immune sensors and triggers the type I IFN response,
disruption of these cellular processes allows the virus to replicate
its genome while minimizing the host innate immune response.
Disruption of these three non-overlapping steps of protein pro-
duction may represent a multi-pronged mechanism that synergis-
tically acts to suppress the host antiviral response (Figure 7F). Spe-
cifically, the IFN response is usually boosted more than 1,000-fold
upon virus detection (through amplification and feedback;
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A B E

C D

Figure 7. NSP8 and NSP9 Inhibit Membrane and Secretory Protein Trafficking
(A) Quantification of HEK293T cells transfected with plasmids co-expressing GFP-tagged NSPs and the NGFR membrane protein. Plotted is the ratio of NGFR to
GFP levels for each condition.
(B) The ratio of puromycin-containing proteins at the cell membrane normalized to GFP expression for each condition.
(C) Quantification of two mRNA reporters containing SL1 fused to GFP (leader-GFP) or NGFR (leader-NGFR) in Vero cells infected with SARS-CoV-2 or mock
infected for 24 h (MOI, 0.1). Plotted is the ratio of leader-NGFR to leader-GFP, binned by increasing amounts of S protein.
(D) Density plot for leader-NGFR to leader-GFP ratios in virally infected Vero cells or mock-treated controls. Replicate conditions were merged for display.
(E) Model of how NSP8/9 act to suppress SRP-dependent protein trafficking upon viral infection.
(F) A model of how SARS-CoV-2 suppresses host immune responses through multi-pronged inhibition of core cellular functions. Cellular mechanisms are shown
in gray and viral mechanisms in red.
Error bars represent standard deviation across independent biological replicates, and dots represent individual values for each replicate; *p < 0.05 and **p < 0.01.

Cell 183, 1325–1339, November 25, 2020 1335

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Figure S4K), but each individual mechanism affects IFN levels 5-
to 10-fold. Accordingly, if each independent mechanism moder-
ately affects IFN levels, the three together may be able to achieve
dramatic suppression of IFN (103 = 1,000-fold). This multi-pronged
mechanism may explain the molecular basis of the potent sup-
pression of IFN observed in patients with severe COVID-19.
IFN is emerging not only as a determinant of disease severity
but also as a potential treatment option (Zhou et al., 2020). Our
work identifies several therapeutic opportunities for boosting
IFN levels upon SARS-CoV-2 infection. For example, disrupting
the interaction between NSP1 and 18S rRNA could allow cells to
detect and respond to viral infection. Because many small-mole-
cule drugs target ribosomal RNAs (Liaud et al., 2019), it may be
possible to develop drugs to block NSP1-18S and other interac-
tions. Additionally, disrupting the 50 viral leader may be a potent
antivirus strategy because it is critical for translation of all viral
proteins. Because SL1 is a structured RNA, it may be possible
to design small molecules that specifically bind this structure
to suppress viral protein production (Hermann, 2016).
Viral suppression of these cellular functions is not exclusive to
the IFN response and also affects other spliced, translated,
secreted, and membrane proteins. Many proteins involved in
anti-viral immunity are spliced and/or membrane anchored or
secreted; for example, MHC class I, critical for antigen presenta-
tion to CD8 T cells at the cell surface of infected cells (Hansen and
Bouvier, 2009). By antagonizing membrane trafficking, SARS-
CoV-2 may prevent viral antigens from being presented on MHC
and allow infected cells to escape T cell recognition and clear-
ance. In this way, interference with these essential cellular pro-
cesses might further aid SARS-CoV-2 to evade the host immune
response.
More generally, we expect that insights gained from the
SARS-CoV-2 protein-RNA binding maps will be critical for
exploring additional viral mechanisms. Specifically, we identified
many other interactions, including highly specific interactions
with mRNAs. For example, NSP12 binds to the JUN mRNA (Fig-
ure S1E), which encodes the critical immune transcription factor
c-Jun, which is activated in response to multiple cytokines and
immune signaling pathways (Weston and Davis, 2007). We also
identified an interaction between NSP9 and the start codon of
the mRNA that encodes COPS5 (Figure 1C), the enzymatic sub-
unit of the COP9 signalosome complex, which regulates protein
homeostasis (Cope and Deshaies, 2003), suggesting that it
might disrupt its translation. Interestingly, COPS5 (also known
as JAB1) is known to bind and stabilize c-Jun protein (Claret
et al., 1996), and several viruses are known to disrupt this protein
(Lungu et al., 2008; Oh et al., 2006; Tanaka et al., 2006). Although
it remains unknown what, if any, role these interactions have in
virus-infected cells, the specificity suggests that they may pro-
vide a selective advantage for virus propagation.
Our results demonstrate that global mapping of RNA binding
by viral proteins could enable rapid characterization of mecha-
nisms of emerging pathogenic RNA viruses.
Limitations of Study
Several limitations of our current study will need to be explored in
future work. (1) Our mapping experiments were performed with
uninfected human cells expressing tagged viral proteins.
1336 Cell 183, 1325–1339, November 25, 2020
Article
Accordingly, it remains possible that our maps may not fully cap-
ture all of the interactions that occur when human cells are in-
fected, such as interactions that occur with virus-induced
RNAs, in specific viral compartments, or that require multiple
viral proteins. (2) Although we characterized the functional and
mechanistic roles of several viral proteins and structural
ncRNAs, we did not explore the roles of viral protein interactions
with mRNAs. (3) How the virus disrupts fundamental cellular pro-
cesses while maintaining its own production is still largely unde-
fined. Although we showed that the 50 leader is sufficient to
relieve translational inhibition by NSP1, we still do not fully under-
stand how this protection occurs and, specifically, how NSP1
might interact with the viral leader or allosterically modulate ribo-
some binding. Similarly, viral membrane proteins are dependent
on trafficking to the ER, and how NSP8/9 might selectively affect
ER translocation of host but not viral proteins remains to be
explored. (4) Although we showed that viral disruption of these
essential cellular functions can suppress IFN, other roles of
host cell shutdown in viral pathogenesis and in suppressing
other aspects of antiviral immunity, including possible roles in
adaptive immune responses, have not been explored.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d RESOURCE AVAILABILITY
B Lead Contact
B Materials Availability
B Data and Code Availability
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
B Cell lines used in this study
B Cell culture conditions
B SARS-CoV-2 Viral Infection strains and conditions
d METHOD DETAILS
B Generation of RNA binding maps
B Antibody Generation
B Microscopy imaging
B Structure modeling
B Recombinant NSP1 production
B In vitro translation assays
B In vivo translation assays
B SUnSET assay
B SUnSET in SARS-CoV-2 infected cells
B Membrane protein reporter experiments
B siRNA experiments for SRP19 and SRP54
B Leader-NGFR measurements
B Membrane SUnSET assay
B Splicing assessment experiments
B Interferon stimulation experiments
B 50 viral leader experiments
B Alignments and phylogeny reconstructions
d QUANTIFICATION AND STATISTICAL ANALYSIS
B Analysis of SARS-CoV-2 viral protein binding to RNA
B Splicing analysis of 5EU data
B Analysis of total RNA in SARS-CoV-2 infected samples

Article
SUPPLEMENTAL INFORMATION
Supplemental Information can be found online at https://doi.org/10.1016/j.
cell.2020.10.004.
ACKNOWLEDGMENTS
We thank Fritz Roth for clones; Marko Jovanovic, Bil Clemons, Shu-ou Shan,
and Jamie Wangen for guidance and discussions; Joanna Jachowicz for ana-
lyses; Shawna Hiley for editing; Inna-Marie Strazhnik for figures; and Scott
Tighe, Pheobe Laaguiby, Roxana del Rio-Guerra, Joyce Oetjen, Philip Eisen-
hauer, Arvind H. Patel, and Arthur Wickenhagen for technical help. This work
was supported by NIH F30HL136080 (to A.K.B.); P20GM125498 and
P30GM118228-04 (to E.A.B.); NIH GM7616-40 and F30CA247447 (to P.B.);
UM1HL120877 and U54GM115516 (to D.M.); R41AI132047 and
U01AI1141997 (to J.W.B.); U01 DA040612 and U01 HL130007 (to M.G.); the
USC MD/PhD Program (to A.K.B.); UCLA-Caltech MSTP (to P.B.); American
Cancer Society PF-17-240-01 (to N.O.); Wellcome Trust 201366/Z/16/Z (to
S.J.R.); the Office of the Vice President for Research at UVM (to J.W.B.); Her-
itage Medical Research Institute (to M.G. and R.V.); and NYSCF, CZI, and Cal-
tech (to M.G.). M.G. is a NYSCF-Robertson Investigator.
AUTHOR CONTRIBUTION
A.K.B. conceived the project and performed protein and 5EU purification,
sequencing, NSP1 and 50 leader flow cytometry, and analyses. M.R.B. devel-
oped and optimized viral protein purification methods, optimized and per-
formed translation assays and splicing assays, and led development and
execution of biochemical assays and functional experiments. E.A.B. led virus
work, designed and performed experiments, and provided guidance and sup-
port regarding analyses, ideas, and discussions of the paper. D.D.H. per-
formed NSP1 translational assays and imaging experiments. L.M.C. per-
formed 50 leader experiments. A.C. performed membrane reporter and
puromycin experiments. P.B. developed 5EU selection and splicing analyses.
N.O., S.A.Q., A.E.L., R.V., and M.G. performed analyses. D.G.S., D.G., G.D.L.,
and S.J.R. generated viral antibodies and performed viral infection. C.L. per-
formed imaging and analysis. J.T., Z.D.M., and M.M.S. performed experi-
ments. R.M.V. provided guidance regarding ribosome and SRP structure
and function. J.W.B. provided guidance and reagents for virus work. D.M.
and M.G. led the project, supervised experiments and analyses, and wrote
the paper.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: June 18, 2020
Revised: August 26, 2020
Accepted: October 2, 2020
Published: October 8, 2020
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